Cell, 2002 Nov 1, 111(3), 369 - 79 Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes; Hassan AH et al.; The functions of the SAGA and SWI/SNF complexes are interrelated and can form stable "epigenetic marks" on promoters in vivo . Here we show that stable promoter occupancy by SWI/SNF and SAGA in the absence of transcription activators requires the bromodomains of the Swi2/Snf2 and Gcn5 subunits, respectively, and nucleosome acetylation . This acetylation can be brought about by either the SAGA or NuA4 HAT complexes . The bromodomain in the Spt7 subunit of SAGA is dispensable for this activity but will anchor SAGA if it is swapped into Gcn5, indicating that specificity of bromodomain function is determined in part by the subunit it occupies . Thus, bromodomains within the catalytic subunits of SAGA and SWI/SNF anchor these complexes to acetylated promoter nucleosomes. Mol Cell, 2002 Oct, 10(4), 935 - 42 Essential role for the SANT domain in the functioning of multiple chromatin remodeling enzymes; Boyer LA et al.; The SANT domain is a novel motif found in a number of eukaryotic transcriptional regulatory proteins that was identified based on its homology to the DNA binding domain of c-myb . Here we show that the SANT domain is essential for the in vivo functions of yeast Swi3p, Ada2p, and Rsc8p, subunits of three distinct chromatin remodeling complexes . We also find that the Ada2p SANT domain is essential for histone acetyltransferase activity of native, Gcn5p-containing HAT complexes . Furthermore, kinetic analyses indicate that an intact SANT domain is required for an Ada2p-dependent enhancement of histone tail binding and enzymatic catalysis by Gcn5p . Our results are consistent with a general role for SANT domains in functional interactions with histone N-terminal tails. Mol Cell, 2002 Oct, 10(4), 925 - 33 Transcriptional inhibition of genes with severe histone h3 hypoacetylation in the coding region; Kristjuhan A et al.; Changes in histone acetylation at promoters correlate with transcriptional activation and repression, but whether acetylation of histones in the coding region of genes is important for transcription is less clear . Here, we show that cells lacking the histone acetyltransferases Gcn5 and Elp3 have widespread and severe histone H3 hypoacetylation in chromatin . Surprisingly, severe hypoacetylation in the promoter does not invariably affect the ability of TBP to bind the TATA element, or transcription of the gene . By contrast, similar hypoacetylation of the coding region correlates with inhibition of transcription, and inhibition correlates better with the overall charge of the histone H3 tail than with hypoacetylation of specific lysine residues . These data provide insights into the effects of histone H3 hypoacetylation in vivo and underscore the importance of the overall charge of the histone tail for transcription. Mol Cell, 2002 Oct, 10(4), 871 - 82 Interplay of TBP inhibitors in global transcriptional control; Chitikila C et al.; The TATA binding protein (TBP) is required for the expression of nearly all genes and is highly regulated both positively and negatively . Here, we use DNA microarrays to explore the genome-wide interplay of several TBP-interacting inhibitors in the yeast Saccharomyces cerevisiae . Our findings suggest the following: The NC2 inhibitor turns down, but not off, highly active genes . Autoinhibition of TBP through dimerization contributes to transcriptional repression, even at repressive subtelomeric regions . The TAND domain of TAF1 plays a primary inhibitory role at very few genes, but its function becomes widespread when other TBP interactions are compromised . These findings reveal that transcriptional output is limited in part by a collaboration of different combinations of TBP inhibitory mechanisms. Przegl Lek, 2002, 59(6), 468 - 71 {Immunologic methods in diagnosis of inflammatory bowel disease}; Macura-Biegun A et al.; Chronic inflammatory bowel disease is included into autoimmune diseases, which is proved by presence of inflammatory lesions, circulating autoantibodies and the deposits of immune complexes in tissues . Coeliac disease (classic and atypical form), Crohn's Disease and ulcerative colitis are considered as chronic autoimmune bowel diseases . The auto-antibodies can be detected with indirect immunofluorescence, ELISA and immunoblotting methods . In coeliac disease the autoantibodies against endomysium (tissue transglutaminase) and antibodies against gliadin are found in patient's sera . The auto-antibody detecting is helpful in establishing diagnosis, controlling gluten-free diet effectiveness and during gluten challenge . In Crohn's disease and ulcerative colitis immunological laboratory tests are helpful to confirm the clinical diagnosis . The following auto-antibodies are tested: against cytoplasm of exocrinal pancreatic cells, and products of these cells, against the neutrophile cytoplasm and against goblet cells . The antibodies against Saccharomyces cerevisiae are also investigated . The clinical relevance of above autoantibodies is not clear, but it is suggested that their presence correlates with exacerbations and severe clinical outcome . In the present study the pictures of autoantibodies from fluorescent microscope were shown. Am J Hum Genet, 2002 Dec, 71(6), 1443 - 9 Epub 2002 Nov 04. A missense mutation (R565W) in cirhin (FLJ14728) in North American Indian childhood cirrhosis; Chagnon P et al.; North American Indian childhood cirrhosis (CIRH1A, or NAIC), a severe autosomal recessive intrahepatic cholestasis described in Ojibway-Cree children from northwestern Quebec, is one of several familial cholestases with unknown molecular etiology . It typically presents with transient neonatal jaundice, in a child who is otherwise healthy, and progresses to biliary cirrhosis and portal hypertension . Clinical and physiological investigations have not revealed the underlying cause of the disease . Currently, liver transplantation is the only effective therapy for patients with advanced disease . We previously identified the NAIC locus by homozygosity mapping to chromosome 16q22 . Here we report that an exon 15 mutation in gene FLJ14728 (alias Cirhin) causes NAIC: c.1741C-->T in GenBank cDNA sequence NM_032830, found in all NAIC chromosomes, changes the conserved arginine 565 codon to a tryptophan, altering the predicted secondary structure of the protein . Cirhin is preferentially expressed in embryonic liver, is predicted to localize to mitochondria, and contains WD repeats, which are structural motifs frequently associated with molecular scaffolds. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14706 - 9 Epub 2002 Nov 04. Transcriptional activating regions target a cyclin-dependent kinase; Ansari AZ et al.; Several yeast activators are phosphorylated by SRB10, a cyclin-dependent kinase associated with the transcriptional machinery . Sites of phosphorylation are found outside the activating region in each case, and the modification has different physiological consequences in different cases . We show here that certain acidic transcriptional activating regions contact SRB10 as assayed both in vivo and in vitro . The interaction evidently positions each activator, as it activates transcription, so that it gets phosphorylated by SRB10, and thus a common mechanism targets disparate substrates to the kinase. Mol Cell Biol, 2002 Dec, 22(23), 8353 - 65 Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair; Qin S et al.; The modification of newly synthesized histones H3 and H4 by type B histone acetyltransferases has been proposed to play a role in the process of chromatin assembly . The type B histone acetyltransferase Hat1p and specific lysine residues in the histone H3 NH(2)-terminal tail (primarily lysine 14) are redundantly required for telomeric silencing . As many gene products, including other factors involved in chromatin assembly, have been found to participate in both telomeric silencing and DNA damage repair, we tested whether mutations in HAT1 and the histone H3 tail were also sensitive to DNA-damaging agents . Indeed, mutations both in specific lysine residues in the histone H3 tail and in HAT1 resulted in sensitivity to methyl methanesulfonate . The DNA damage sensitivity of the histone H3 and HAT1 mutants was specific for DNA double-strand breaks, as these mutants were sensitive to the induction of an exogenous restriction endonuclease, EcoRI, but not to UV irradiation . While histone H3 mutations had minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in the recombinational repair of DNA double-strand breaks . Epistasis analysis indicates that the histone H3 and HAT1 mutants may influence DNA double-strand break repair through Asf1p-dependent chromatin assembly. Mol Cell Biol, 2002 Dec, 22(23), 8292 - 301 Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning; Andrulis ED et al.; A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1 . A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7 . Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected . On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants . Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262) . When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo . Here, both phenomena were shown to require ESC1 . Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1 . Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing . GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes . Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery. Mol Cell Biol, 2002 Dec, 22(23), 8254 - 66 Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation; Libri D et al.; Several aspects of eukaryotic mRNA processing are linked to transcription . In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional elongation . Indeed, we find that a pool of heat shock HSP104 transcripts are 3'-end truncated in THO complex mutant as well as sub2 mutant backgrounds . Surprisingly, however, this defect can be suppressed by deletion of the 3'-5' exonuclease Rrp6p . This indicates that incomplete RNAs result from nuclear degradation rather than from a failure to efficiently elongate transcription . RNAs that are not degraded are retained at the transcription site in a Rrp6p-dependent manner . Interestingly, the addition of a RRP6 deletion to sub2 or to THO complex mutants shows a strong synthetic growth phenotype, suggesting that the failure to retain and/or degrade defective mRNAs is deleterious . mRNAs produced in the 3'-end processing mutants rna14-3 and rna15-2, as well as an RNA harboring a 3' end generated by a self-cleaving hammerhead ribozyme, are also retained in Rrp6p-dependent transcription site foci . Taken together, our results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p. Mol Cell Biol, 2002 Dec, 22(23), 8241 - 53 Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p; Zenklusen D et al.; Yra1p/REF participates in mRNA export by recruiting the export receptor Mex67p to messenger ribonucleoprotein (mRNP) complexes . Yra1p also binds Sub2p, a DEAD box ATPase/RNA helicase implicated in splicing and required for mRNA export . We identified genetic and physical interactions between Yra1p, Sub2p, and Hpr1p, a protein involved in transcription elongation whose deletion leads to poly(A)(+) RNA accumulation in the nucleus . By chromatin immunoprecipitation (ChIP) experiments, we show that Hpr1p, Sub2p, and Yra1p become associated with active genes during transcription elongation and that Hpr1p is required for the efficient recruitment of Sub2p and Yra1p . The data indicate that transcription and export are functionally linked and that mRNA export defects may be due in part to inefficient loading of essential mRNA export factors on the growing mRNP . We also identified functional interactions between Yra1p and the exosome components Rrp45p and Rrp6p . We show that yra1, sub2, and Deltahpr1 mutants all present defects in mRNA accumulation and that deletion of RRP6 in yra1 mutants restores normal mRNA levels . The data support the hypothesis that an exosome-dependent surveillance mechanism targets improperly assembled mRNPs for degradation. Mol Cell Biol, 2002 Dec, 22(23), 8215 - 25 NuA4 subunit Yng2 function in intra-S-phase DNA damage response; Choy JS et al.; While regulated transcription requires acetylation of histone N-terminal tails to promote an open chromatin conformation, a similar role for histone acetylation in DNA replication and/or repair remains to be established . Cells lacking the NuA4 subunit Yng2 are viable but critically deficient for genome-wide nucleosomal histone H4 acetylation . We found that yng2 mutants are specifically sensitized to DNA damage in S phase induced by cdc8 or cdc9 mutations, hydroxyurea, camptothecin, or methylmethane sulfonate (MMS) . In yng2, MMS treatment causes a persistent Mec1-dependent intra-S-phase checkpoint delay characterized by slow DNA repair . Restoring H4 acetylation with the histone deacetylase inhibitor trichostatin A promotes checkpoint recovery . In turn, mutants lacking the histone H3-specific acetyltransferase GCN5 are similarly sensitive to intra-S-phase DNA damage . The inviability of gcn5 yng2 double mutants suggests overlapping roles for H3 and H4 acetylation in DNA replication and repair . Paradoxically, haploid yng2 mutants do not tolerate mutations in genes important for nonhomologous end joining repair yet remain proficient for homologous recombination . Our results implicate nucleosomal histone acetylation in maintaining genomic integrity during chromosomal replication. Mol Cell Biol, 2002 Dec, 22(23), 8114 - 21 Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay; Lykke-Andersen J; Decapping is a key step in general and regulated mRNA decay . In Saccharomyces cerevisiae it constitutes a rate-limiting step in the nonsense-mediated decay pathway that rids cells of mRNAs containing premature termination codons . Here two human decapping enzymes are identified, hDcp1a and hDcp2, as well as a homolog of hDcp1a, termed hDcp1b . Transiently expressed hDcp1a and hDcp2 proteins localize primarily to the cytoplasm and form a complex in human cell extracts . hDcp1a and hDcp2 copurify with decapping activity, an activity sensitive to mutation of critical hDcp residues . Importantly, coimmunoprecipitation assays demonstrate that hDcp1a and hDcp2 interact with the nonsense-mediated decay factor hUpf1, both in the presence and in the absence of the other hUpf proteins, hUpf2, hUpf3a, and hUpf3b . These data suggest that a human decapping complex may be recruited to mRNAs containing premature termination codons by the hUpf proteins. Plant Cell, 2002 Nov, 14(11), 2915 - 27 Regulation of the pollen-specific actin-depolymerizing factor LlADF1; Allwood EG et al.; Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved . We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1 . Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation . Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains . This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth . We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by approximately 60% . Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes . Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy. J Neurosci, 2002 Nov 1, 22(21), 9490 - 501 Functional dissection of neuroanatomical loci regulating ethanol sensitivity in Drosophila; Rodan AR et al.; Ethanol has complex but similar effects on behavior in mammals and the fruit fly Drosophila melanogaster . In addition, genetic and pharmacological approaches have implicated the cAMP pathway in the regulation of ethanol-induced behaviors in both flies and rodents . Here we examine the neuroanatomical loci that modulate ethanol sensitivity in Drosophila by targeting the expression of an inhibitor of cAMP-dependent protein kinase (PKA) to specific regions in the fly's brain . Expression of the inhibitor in most brain regions or in muscle has no effect on behavior . In contrast, inhibition of PKA in a relatively small number of cells, possibly neurosecretory cells, in the fly's brain is sufficient to decrease sensitivity to the incoordinating effects of ethanol . Additional brain areas are, however, also involved . The mushroom bodies, brain structures where cAMP signaling is required for olfactory classical conditioning, are dispensable for the regulation of ethanol sensitivity . Finally, different behavioral effects of ethanol, motor incoordination and sedation, appear to be regulated by PKA function in distinct brain regions . We conclude that the regulation of ethanol-induced behaviors by PKA involves complex interactions among groups of cells that mediate either increased or reduced sensitivity to the acute intoxicating effects of ethanol. J Biol Chem, 2003 Jan 10, 278(2), 896 - 907 Epub 2002 Nov 03. Identification of allosteric peptide agonists of CXCR4; Sachpatzidis A et al.; The chemokine receptor CXCR4 is a co-receptor for T-tropic strains of HIV-1 . A number of small molecule antagonists of CXCR4 are in development but all are likely to lead to adverse effects due to the physiological function of CXCR4 . To prevent these complications, allosteric agonists may be therapeutically useful as adjuvant therapy in combination with small molecule antagonists . A synthetic cDNA library coding for 160,000 different SDF-based peptides was screened for CXCR4 agonist activity in a yeast strain expressing a functional receptor . Peptides that activated CXCR4 in an autocrine manner induced colony formation . Two peptides, designated RSVM and ASLW, were identified as novel agonists that are insensitive to the CXCR4 antagonist AMD3100 . In chemotaxis assays using the acute lymphoblastic leukemia cell line CCRF-CEM, RSVM behaves as a partial agonist and ASLW as a superagonist . The superagonist activity of ASLW may be related to its inability to induce receptor internalization . In CCRF-CEM cells, the two peptides are also not inhibited by another CXCR4 antagonist, T140, or the neutralizing monoclonal antibodies 12G5 and 44717.111 . These results suggest that alternative agonist-binding sites are present on CXCR4 that could be screened to develop molecules for therapeutic use. FEBS Lett, 2002 Nov 6, 531(2), 259 - 64 A split-ubiquitin-based assay detects the influence of mutations on the conformational stability of the p53 DNA binding domain in vivo; Johnsson N; Many mutations in the human tumor suppressor p53 affect the function of the protein by destabilizing the structure of its DNA binding domain . To monitor the effects of those mutations in vivo the stability of the DNA binding domain of p53 and some of its mutants was investigated with the split-ubiquitin (split-Ub) method . The split-Ub-derived in vivo data on the relative stability of the mutants roughly correlate with the quantitative data from in vitro denaturation experiments as reported in the literature . A variation of this assay allows visualizing the difference in stability between the wild-type p53 core and the mutant p53(V143A) by a simple growth assay. J Mol Biol, 2002 Nov 8, 323(5), 835 - 43 A novel two-step mechanism for removal of a mitochondrial signal sequence involves the mAAA complex and the putative rhomboid protease Pcp1; Esser K et al.; The yeast protein cytochrome c peroxidase (Ccp1) is nuclearly encoded and imported into the mitochondrial intermembrane space, where it is involved in degradation of reactive oxygen species . It is known, that Ccp1 is synthesised as a precursor with a N-terminal pre-sequence, that is proteolytically removed during transport of the protein . Here we present evidence for a new processing pathway, involving novel signal peptidase activities . The mAAA protease subunits Yta10 (Afg3) and Yta12 (Rca1) were identified both to be essential for the first processing step . In addition, the Pcp1 (Ygr101w) gene product was found to be required for the second processing step, yielding the mature Ccp1 protein . The newly identified Pcp1 protein belongs to the rhomboid-GlpG superfamily of putative intramembrane peptidases . Inactivation of the protease motifs in mAAA and Pcp1 blocks the respective steps of proteolysis . A model of coupled Ccp1 transport and N-terminal processing by the mAAA complex and Pcp1 is discussed . Similar processing mechanisms may exist, because the mAAA subunits and the newly identified Pcp1 protein belong to ubiquitous protein families. J Mol Biol, 2002 Nov 8, 323(5), 811 - 22 Digging deep for ancient relics: a survey of protein motifs in the intergenic sequences of four eukaryotic genomes; Zhang ZL et al.; We have examined conserved protein motifs in the non-coding, intergenic regions ("pseudomotif patterns") and surveyed their occurrence in the fly, worm, yeast and human genomes (chromosomes 21 and 22 only) . To identify these patterns, we masked out annotated genes, pseudogenes and repeat regions from the raw genomic sequence and then compared the remaining sequence, in six-frame translation, against 1319 patterns from the PROSITE database . For each pseudomotif pattern, the absolute number of occurrences is not very informative unless compared against a statistical expectation; consequently, we calculated the expected occurrence of each pattern using a Poisson model and verified this with simulations . Using a p-value cut-off of 0.01, we found 67 pseudomotif patterns over-represented in fly intergenic regions, 34 in worm, 21 in human and six in yeast . These include the zinc finger, leucine zipper, nucleotide-binding motif and EGF domain . Many of the over-represented patterns were common to two or more organisms, but there were a few that were unique to specific ones . Furthermore, we found more over-represented patterns in the fly than in the worm, although the fly has fewer pseudogenes . This puzzling observation can be explained by a higher deletion rate in the fly genome . We also surveyed under-represented patterns, finding 23 in the fly, 12 in the worm, 18 in human and two in yeast . If intergenic sequences were truly random, we would expect an equal number of over and under-represented patterns . The fact that for each organism the number of over-represented patterns is greater than the number of under-represented ones implies that a fraction of the intergenic regions consist of ancient protein fragments that, due to accumulated disablements, have become unrecognizable by conventional techniques for gene and pseudogene identification . Moreover, we find that in aggregate the over-represented pseudomotif patterns occupy a substantial fraction of the intergenic regions . Further information is available at http://pseudogene.org Trends Biochem Sci, 2002 Nov, 27(11), 580 - 5 Nuclear export of ribosomal subunits; Johnson AW et al.; The partitioning of cells by a nuclear envelope ensures that precursors of ribosomes do not interact prematurely with other components of the translation machinery . Ribosomal subunits are assembled in nucleoli and exported to the cytoplasm in a CRM1/Ran-GTP-dependent fashion . Export of the large (60S) subunit requires a shuttling adaptor protein, NMD3, which binds to mature, correctly folded subunits . Immature or defective particles do not bind NMD3 and thus are excluded from the export pathway . This structural proofreading is extended into the cytoplasm, where it is believed that several energy-requiring steps release shuttling factors from the subunit, allowing it to function in translation. Trends Biochem Sci, 2002 Nov, 27(11), 572 - 9 Telomerase: biochemical considerations for enzyme and substrate; Kelleher C et al.; Telomerase extends chromosome ends by iterative reverse transcription of its RNA template . Following the addition of each telomeric repeat, the RNA template and the telomeric substrate reset their relative position in the active site provided by the telomerase reverse transcriptase (TERT) . This step might require the formation of guanine-rich secondary structures in the nascent product . Results from numerous studies begin to delineate TERT sub-domains that orchestrate these events and support the model of cooperative action between distinct active sites within telomerase multimers . Natural telomere substrates are protein-DNA complexes that show an asymmetry between the two ends of a chromosome, possibly reflecting their differential mode of replication. Biochemistry, 2002 Nov 12, 41(45), 13403 - 9 Mutational analysis of baculovirus phosphatase identifies structural residues important for triphosphatase activity in vitro and in vivo; Martins A et al.; Baculovirus phosphatase (BVP) and mammalian capping enzyme (Mce1) are members of the RNA triphosphatase branch of the cysteine phosphatase superfamily . Although RNA triphosphatases have a core alpha/beta fold similar to other cysteine phosphatases, there is little conservation of primary structure outside of the cysteine-containing P-loop motif, HCxxxxxR(S/T), that comprises the active site . However, there is extensive primary structure conservation between members of the RNA triphosphatase branch, whether from cellular or viral sources and whether they are bifunctional capping enzymes such as Mce1 or monofunctional RNA phosphatases such as BVP . To evaluate the functional significance of such sequence conservation, we performed a mutational analysis of 14 residues of BVP . We identified three side chains (Trp6, Lys25, and Arg153) as essential for triphosphatase activity in vitro, i.e., W6A, K25A, and R153A were <0.1% as active as wild-type BVP, and were unable to complement a yeast RNA triphosphatase null mutant in vivo . Six other BVP residues (Thr62, Tyr67, Tyr68, Lys82, Glu158, and Arg159) were deemed functionally important, i.e., Ala mutations reduced triphosphatase activity to <20% of wild-type . On the basis of the locations of the equivalent amino acids in the Mce1 crystal structure, we surmise that the essential/important BVP residues ensure proper conformation of the catalytic P-loop (e.g., Arg153 and Tyr68) or other elements of the tertiary structure . Our results highlight a conserved Trp6-Lys25 pi-cation pair essential for BVP function. Biochemistry, 2002 Nov 12, 41(45), 13395 - 402 Interaction of human DNA topoisomerase II alpha with DNA: quantification by surface plasmon resonance; Renodon-Corniere A et al.; DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double-stranded DNA segment and transporting it through another duplex . Surface plasmon resonance (SPR) was used to characterize interactions of human topoisomerase II alpha with different topological forms of DNA . Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II alpha was determined to be 0.16 nM . The affinity was not affected by the absence of ATP or the presence of the bisdioxopiperazine catalytic inhibitor ICRF-187 . Besides, similar affinities were found for several bisdioxopiperazine-resistant mutant enzymes . These results suggest that the mechanism of topoisomerase II alpha inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with the enzyme . SPR was also adapted to measure levels of the closed clamp form of topoisomerase II present on DNA . As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but not on linear DNA . Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, or a non-hydrolyzable analogue of ATP . In the presence of ATP and ICRF-187, several bisdioxopiperazine-resistant mutant enzymes failed to form detectable levels of stable closed clamp . Interestingly, a mutant of human topoisomerase II alpha with an altered active site tyrosine showed lower levels of closed clamp formation . In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNA substrate and (2) quantify the enzyme's closed clamp formation under varying circumstances. Genome, 2002 Oct, 45(5), 804 - 11 Expression and genomic organization of the zebrafish chaperonin gene complex; Martin CC et al.; Chaperonin 10 and chaperonin 60 monomers exist within the multimeric mitochondrial chaperonin folding complex with a stoichiometry of 2:1 . This complex is located in the mitochondrial matrix, where it aids in the folding and acquisition of the tertiary structure of proteins . We have previously isolated the cpn10 cDNA in zebrafish (Danio rerio), and demonstrated that it is ubiquitously expressed during embryonic development and transcriptionally upregulated after exposure to heat shock . In the present study, we have isolated a cDNA encoding chaperonin 60 (cpn60) from zebrafish, and have shown that it is similarly expressed uniformly and ubiquitously throughout early embryonic development of zebrafish . Upregulation of cpn60 expression was also observed after exposure of zebrafish embryos to a heat shock of 1 h at 37 degrees C compared with control embryos raised at 27 degrees C . The induction of the cpn60 heat shock response was greatest after 1 h of heat shock, whereas significant decreases of cpn60 mRNA were observed within 2 h following a return to 27 degrees C . We subsequently isolated genomic DNA sequences for both of these genes, and show that they are also arranged in a head-to-head organization and share a common bidirectional promoter that contains a single heat shock element (HSE) . Our database analysis shows that this head-to-head organization is also found in human (Homo sapiens), rat (Rattus norvegicus), pufferfish (Fugu rubripes), and Caenorhabditis elegans, but not in Drosophila or yeast (Saccharomyces cerevisiae) . The data suggest that the genomic organization of the cpn gene complex has been conserved across the vertebrates. J Biomed Inform, 2002 Feb, 35(1), 25 - 36 Visualization and evaluation of clusters for exploratory analysis of gene expression data; Kim JH et al.; Clustering algorithms have been shown to be useful to explore large-scale gene expression profiles . Visualization and objective evaluation of clusters are two important considerations when users are selecting different clustering algorithms, but they are often overlooked . The developments of a framework and software tools that implement comprehensive data visualization and objective measures of cluster quality are crucial . In this paper, we describe a theoretical framework and formalizations for consistently developing clustering algorithms . A new clustering algorithm was developed within the proposed framework . We demonstrate that a theoretically sound principle can be uniformly applied to the developments of cluster-optimization function, comprehensive data-visualization strategy, and objective cluster-evaluation measures as well as actual implementation of the principle . Cluster consistency and quality measures of the algorithm are rigorously evaluated against those of popular clustering algorithms for gene expression data analysis (K-means and self-organizing maps), in four data sets, yielding promising results. Rapid Commun Mass Spectrom, 2002, 16(22), 2115 - 23 Amino acid residue specific stable isotope labeling for quantitative proteomics; Zhu H et al.; Various stable isotope labeling (SIL) techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry (MS) . We have developed a mass-tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residue-specific manner during cell growth . In this study, we further extend this residue-specific SIL approach to the accurate quantitation of protein abundances in different cell populations . For proteins whose expression levels are the same in cells grown in the normal and labeled media, the relative areas of the normal (light) and labeled (heavy) isotopic peaks are linearly correlated with the cells mixing ratios . This approach was first used to determine the effect of the zinc-responsive transcription factor Zap1 on the yeast proteome . Ten protein spots from a PAGE gel were chosen randomly and their differential protein expression levels in wild-type and zap1delta cells were readily determined by the isotopic ratio . Methionine synthase (Met6) was identified to be up-regulated more than four times in the zap1delta mutant strain whereas the expression level of other nine proteins remained unchanged . Further, we applied this strategy to study the cellular response to radiation in human skin fibroblast cells . Analyzing one protein band randomly selected from SDS-PAGE, the expression level of a novel protein was found to increase two-fold in response to radiation whereas the expression level of a control protein remained unchanged . This strategy is generally applicable using any particular type of amino acid as the labeling precursors for accurate quantitation of protein relative abundances. J Clin Endocrinol Metab, 2002 Nov, 87(11), 5185 - 90 Thyroid hormone action is disrupted by bisphenol A as an antagonist; Moriyama K et al.; Bisphenol A (BPA), a monomer of polycarbonate plastics, has been shown to possess estrogenic properties and act as an agonist for the estrogen receptors . Although an epidemiologically based investigation has suggested that some chemicals could disrupt thyroid function in animals, the effects on thyroid hormone receptors (TRs) are unknown . We show here that BPA inhibits TR-mediated transcription by acting as an antagonist . In the transient gene expression experiments, BPA suppressed transcriptional activity that is stimulated by thyroid hormone (T(3)) in a dose-dependent manner . The inhibitory effects were observed in the presence of physiological concentrations of T(3) . In contrast, in the case of negatively regulated TSHalpha promoter, BPA activated the gene transcription that is suppressed by T(3) . To elucidate possible mechanisms of the antagonistic action of BPA, the effects on T(3) binding and cofactor interaction with TR were examined . The K(i) value for BPA was 200 micro M when assessed by inhibition of {(125)I}T(3) binding to rat hepatic nuclear TRs . In a mammalian two-hybrid assay, BPA recruited the nuclear corepressor to the TR . These results suggest that BPA could displace T(3) from the TR and recruit a transcriptional repressor, resulting in gene suppression . This is the first report that BPA can antagonize T(3) action at the transcriptional level . BPA may disrupt the function of various types of nuclear hormone receptors and their cofactors to disturb our internal hormonal environment. Genes Dev, 2002 Nov 1, 16(21), 2792 - 9 Promoter proximal splice sites enhance transcription; Furger A et al.; Reconstruction of a gene with its introns removed results in reduced levels of cytoplasmic mRNA . This is partly explained by introns promoting the export of mRNA through coupling splicing to nuclear export processes . However, we show here that splicing signals can have a direct role in enhancing gene transcription . Removal of promoter proximal splice signals from a mammalian gene or the excision of introns from two different yeast genes results in a marked reduction in levels of nascent transcription, based on both nuclear run-on and direct image analysis . This further establishes that mRNA processing and transcription are tightly coupled mechanisms. Genes Dev, 2002 Nov 1, 16(21), 2761 - 6 Intron status and 3'-end formation control cotranscriptional export of mRNA; Lei EP et al.; Messenger RNA export factors are recruited to genes in a transcription-dependent manner . To ascertain the mechanism of this process, we show that RNA polymerase II transcription is sufficient to recruit the Saccharomyces cerevisiae hnRNP protein Npl3 to a gene independent of RNA sequence . In contrast, the cotranscriptional recruitment of the RNA-binding protein Yra1 is dependent on pre-mRNA processing . Yra1 associates with introns of intron-containing genes in a splicing-dependent manner . Conversely, Yra1 recruitment to genes without introns is not dependent on splicing . Finally, 3'-end formation is required for Yra1 recruitment to genes regardless of intron status. Genes Dev, 2002 Nov 1, 16(21), 2755 - 60 A novel mitochondrial protein, Tar1p, is encoded on the antisense strand of the nuclear 25S rDNA; Coelho PS et al.; In eukaryotes, it is widely assumed that genes coding for proteins and structural RNAs do not overlap . Using a transposon-tagging strategy to globally analyze the Saccharomyces cerevisiae genome for expressed genes, we identified multiple insertions in an open reading frame that is contained fully within and transcribed antisense to the 25S rRNA gene in the nuclear rDNA repeat region on Chromosome XII . Expression of this gene, TAR1 (Transcript Antisense to Ribosomal RNA), can be detected at the RNA and protein levels, and the primary sequence of the corresponding 124-amino-acid protein is conserved in several yeast species . Tar1p was found to localize to mitochondria, and overexpression of the protein suppresses the respiration-deficient petite phenotype of a point mutation in mitochondrial RNA polymerase that affects mitochondrial gene expression and mtDNA stability . These findings indicate that coding information for protein and structural RNAs can overlap, raising issues regarding the coevolution of such complex genes, and also suggest that rDNA transcription and mitochondrial function are coordinately regulated in eukaryotic cells. Biophys J, 2002 Nov, 83(5), 2801 - 11 Numerical study of the entropy loss of dimerization and the folding thermodynamics of the GCN4 leucine zipper; Vinals J et al.; A lattice-based model of a protein and the Monte Carlo simulation method are used to calculate the entropy loss of dimerization of the GCN4 leucine zipper . In the representation used, a protein is a sequence of interaction centers arranged on a cubic lattice, with effective interaction potentials that are both of physical and statistical nature . The Monte Carlo simulation method is then used to sample the partition functions of both the monomer and dimer forms as a function of temperature . A method is described to estimate the entropy loss upon dimerization, a quantity that enters the free energy difference between monomer and dimer, and the corresponding dimerization reaction constant . As expected, but contrary to previous numerical studies, we find that the entropy loss of dimerization is a strong function of energy (or temperature), except in the limit of large energies in which the motion of the two dimer chains becomes largely uncorrelated . At the monomer-dimer transition temperature we find that the entropy loss of dimerization is approximately five times smaller than the value that would result from ideal gas statistics, a result that is qualitatively consistent with a recent experimental determination of the entropy loss of dimerization of a synthetic peptide that also forms a two-stranded alpha-helical coiled coil. Biophys J, 2002 Nov, 83(5), 2726 - 32 Thin filament regulation and ionic interactions between the N-terminal region in actin and troponin; Wong WW et al.; The N-terminal region in actin has been shown to interact with both myosin and troponin (Tn) during the cross-bridge cycle and in regulation . To study the role of this region in regulation, we used yeast actin mutants with increased and decreased numbers of acidic residues . The mutants included D24A/D25A, with Asp(24) and Asp(25) replaced with alanines; DNEQ, with the substitution of Asp(2) and Glu(4) with their amide analogs; and 4Ac, with Glu(3) and Asp(4) inserted in lieu of Ser(3) . In the in vitro motility assay, using reconstituted regulated thin filaments, the sliding speeds of DNEQ, D24A/D25A, and 4Ac were similar at all pCa values . Thus, Ca(2+)-sensitivity of the thin filaments and the inhibitory function of TnI appear to be insensitive to changes in charge (+/-2) at the N-terminus of actin, suggesting little, if any, role of that actin region in regulation . A Ca(2+)-independent conformational change in that region was detected upon troponin binding to actin-Tm via an increase in the fluorescence of a pyrene probe attached to another yeast actin mutant that we used (Cys(1)). J Biotechnol, 2003 Jan 9, 100(1), 55 - 64 Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins; Cunha MT et al.; Genetic engineering was integrated with the production and purification of Fusarium solani pisi cutinases, in order to obtain the highest amount of enzyme activity units, after purification . An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular cutinases expressed in Saccharomyces cerevisiae . The production/extraction process was evaluated regarding cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS . The proteins studied were cutinase wild type and two fusion proteins of cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)(2) and (WP)(4) . The (WP)(4) fusion protein enabled a 300-fold increase of the cutinase partition coefficient when comparing to the wild type . However, the secretion of the fusion proteins was lower than of the wild type cutinase secretion . A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC) . The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth . The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy . Considering the integrated process, the cutinase-(WP)(2) proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)(4) fusion proteins, respectively. Curr Opin Chem Biol, 2002 Oct, 6(5), 689 - 95 Imaging optical sensor arrays; Walt DR; Imaging optical fibres have been etched to prepare microwell arrays . These microwells have been loaded with sensing materials such as bead-based sensors and living cells to create high-density sensor arrays . The extremely small sizes and volumes of the wells enable high sensitivity and high information content sensing capabilities. Curr Opin Chem Biol, 2002 Oct, 6(5), 666 - 75 Analytical aspects of mass spectrometry and proteomics; Mo W et al.; Mass spectrometry plays an essential role in proteomics analysis and research . In recent years, it has been increasingly recognized that a key to proteomics using mass spectrometry relies not only on the instrument itself, but also on the analytical strategies and front-end sample-handling techniques . The advances of separations and mass spectrometry are having an increasing impact on the discovery of disease biomarkers and the understanding of cellular processes. Arch Biochem Biophys, 2002 Nov 15, 407(2), 189 - 95 The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23; Meyn MA 3rd et al.; Properly regulated cyclin proteolysis is critical for normal cell cycle progression . A nine-amino acid peptide motif called the destruction box (D box) is present at the N terminus of the yeast mitotic cyclins . This short sequence is required for cyclin ubiquitination and subsequent proteolysis . The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 required for cyclin ubiquitination . We have tested the D box of five mitotic cyclins for interaction with six APC/C subunits . The APC/C subunit Cdc23, but not five other subunits tested, interacted by two-hybrid analysis with the N terminus of wild-type Clb2 . None of these subunits interacted with the N termini of the cyclins Clb1, Clb3, or Clb5 . Mutations in the D box sequences of Clb2 inhibited interaction with Cdc23 both in vivo and in vitro . Our results provide the first evidence for a direct interaction between an APC/C substrate (Clb2) and an APC/C subunit (Cdc23). Arch Biochem Biophys, 2002 Nov 15, 407(2), 143 - 51 Phosphatidylinositol 3,5-bisphosphate: metabolism and function; Cooke FT; Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) is the most recently discovered PtdInsP(2) isomer . It is likely that PtdIns(3,5)P(2) is ubiquitous to eukaryotes, and that it performs a number of important cellular functions, including vacuolar homeostasis, retrograde trafficking from the vacuole, and protein sorting at the multivesicular body . This review describes the metabolism of PtdIns(3,5)P(2) and discusses the potential functions for this lipid. Anal Biochem, 2002 Oct 15, 309(2), 196 - 9 Size distribution measurement of vesicles by atomic force microscopy; Kanno T et al.; Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems . When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body . Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal . Therefore atomic force microscopy was attempted as another size measurement technique . After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure . The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle . At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate . The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle . Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies. BMC Bioinformatics . 2002 Nov 01;3(1):34. Automated modelling of signal transduction networks; Steffen M et al.; BACKGROUND: Intracellular signal transduction is achieved by networks of proteins and small molecules that transmit information from the cell surface to the nucleus, where they ultimately effect transcriptional changes . Understanding the mechanisms cells use to accomplish this important process requires a detailed molecular description of the networks involved . RESULTS: We have developed a computational approach for generating static models of signal transduction networks which utilizes protein-interaction maps generated from large-scale two-hybrid screens and expression profiles from DNA microarrays . Networks are determined entirely by integrating protein-protein interaction data with microarray expression data, without prior knowledge of any pathway intermediates . In effect, this is equivalent to extracting subnetworks of the protein interaction dataset whose members have the most correlated expression profiles . CONCLUSION: We show that our technique accurately reconstructs MAP Kinase signaling networks in Saccharomyces cerevisiae . This approach should enhance our ability to model signaling networks and to discover new components of known networks . More generally, it provides a method for synthesizing molecular data, either individual transcript abundance measurements or pairwise protein interactions, into higher level structures, such as pathways and networks. EMBO J, 2002 Nov 1, 21(21), 5886 - 98 Direct eIF2-eIF3 contact in the multifactor complex is important for translation initiation in vivo; Valasek L et al.; Translation initiation factor 3 (eIF3) of Saccharo myces cerevisiae forms a multifactor complex (MFC) with eIFs 1, 2, 5 and Met-tRNA(i)(Met) . We previously constructed a subunit interaction model for the MFC . Here we incorporated affinity tags into the three largest eIF3 subunits (eIF3a/TIF32, eIF3b/PRT1 and eIF3c/NIP1) and deleted predicted binding domains in each tagged protein . By characterizing the mutant subcomplexes, we confirmed all key predictions of our model and uncovered new interactions of NIP1 with PRT1 and of TIF32 with eIF1 . In addition to the contact between eIF2 and the N-terminal domain (NTD) of NIP1 bridged by eIF5, the C-terminal domain (CTD) of TIF32 binds eIF2 directly and is required for eIF2-eIF3 association in vivo . Overexpressing a CTD-less form of TIF32 exacerbated the initiation defect of an eIF5 mutation that weakens the NIP1-eIF5-eIF2 connection . Thus, the two independent eIF2-eIF3 contacts have additive effects on translation in vivo . Overexpressing the NIP1-NTD sequestered eIF1-eIF5-eIF2 in a defective subcomplex that derepressed GCN4 translation, providing the first in vivo evidence that association with eIF3 promotes binding of eIF2 and Met-tRNA(i)(Met) to 40S ribosomes. EMBO J, 2002 Nov 1, 21(21), 5843 - 52 The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores; Fischer T et al.; Yra1p and Sub2p are components of the TREX complex, which couples transcription elongation with nuclear export of mRNAs . Here, we report a genetic interaction between Yra1p and a conserved protein Sac3p, which previously was found to interact with Sub2p . In vivo, Sac3p forms a stable complex with Thp1p, which was reported to function in transcription elongation . In addition, Sac3p binds to the mRNA exporter Mex67p-Mtr2p and requires the nucleoporin Nup1p to dock at the nuclear side of the nuclear pore complex (NPC) . Significantly, mutations in Sac3p or Thp1p lead to strong mRNA export defects . Taken together, our data suggest that the novel Sac3p-Thp1p complex functions by docking the mRNP to specific nucleoporins at the nuclear entrance of the NPC. EMBO J, 2002 Nov 1, 21(21), 5833 - 42 Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner; Miyamoto Y et al.; A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex . In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay . A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus . Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta . These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner . Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae . Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments . This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS . These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis. EMBO J, 2002 Nov 1, 21(21), 5775 - 86 Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function; Schlisio S et al.; To explore mechanisms for specificity of function within the family of E2F transcription factors, we have identified proteins that interact with individual E2F proteins . A two-hybrid screen identified RYBP (Ring1- and YY1-binding protein) as a protein that interacts specifically with the E2F2 and E2F3 family members, dependent on the marked box domain in these proteins . The Cdc6 promoter contains adjacent E2F- and YY1-binding sites, and both are required for promoter activity . In addition, YY1 and RYBP, in combination with either E2F2 or E2F3, can stimulate Cdc6 promoter activity synergistically, dependent on the marked box domain of E2F3 . Using chromatin immunoprecipitation assays, we show that both E2F2 and E2F3, as well as YY1 and RYBP, associate with the Cdc6 promoter at G(1)/S of the cell cycle . In contrast, we detect no interaction of E2F1 with the Cdc6 promoter . We suggest that the ability of RYBP to mediate an interaction between E2F2 or E2F3 and YY1 is an important component of Cdc6 activation and provides a basis for specificity of E2F function. EMBO J, 2002 Nov 1, 21(21), 5721 - 32 Structure of the sporulation-specific transcription factor Ndt80 bound to DNA; Lamoureux JS et al.; Progression through the middle phase of sporulation in Saccharomyces cerevisiae is promoted by the successful completion of recombination at the end of prophase I . Completion of meiotic recombination allows the activation of the sporulation-specific transcription factor Ndt80, which binds to a specific DNA sequence, the middle sporulation element (MSE), and activates approximately 150 genes to enable progression through meiosis . Here, we isolate the DNA-binding domain of Ndt80 and determine its crystal structure both free and in complex with an MSE-containing DNA . The structure reveals that Ndt80 is a member of the Ig-fold family of transcription factors . The structure of the DNA-bound form, refined at 1.4 A, reveals an unexpected mode of recognition of 5'-pyrimidine- guanine-3' dinucleotide steps by arginine residues that simultaneously recognize the 3'-guanine base through hydrogen bond interactions and the 5'-pyrimidine through stacking/van der Waals interactions . Analysis of the DNA-binding affinities of MSE mutants demonstrates the central importance of these interactions, and of the AT-rich portion of the MSE . Functional similarities between Ndt80 and the Caenorhabditis elegans p53 homolog suggest an evolutionary link between Ndt80 and the p53 family. J Biol Chem, 2003 Jan 10, 278(2), 1075 - 85 Epub 2002 Oct 30. Isolation and characterization of TgVP1, a type I vacuolar H+-translocating pyrophosphatase from Toxoplasma gondii . The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor; Drozdowicz YM et al.; Here we report the isolation and characterization of a type I vacuolar-type H(+)-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation . The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category . Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T . gondii . Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K(+)-activated pyrophosphate (PP(i)) hydrolysis and PP(i)-energized H(+) translocation . Immunofluorescence microscopy of both free and intracellular T . gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme . Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow . Although treatment of T . gondii with AMDP concentrations as high as 100 microm had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I(50) for the inhibition of TgVP1 activity in vitro (0.9 microm) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures . A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion. Blood, 2003 Mar 15, 101(6), 2328 - 34 Epub 2002 Oct 31. Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia; Molina-Arcas M et al.; Nucleoside derivatives are currently used in the treatment of hematologic malignancies . Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed . We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes . We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity . Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed . Fludarabine accumulation in CLL cells was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL. Nat Genet, 2002 Nov, 32(3), 370 - 7 Epub 2002 Oct 15. Chromosomal gradient of histone acetylation established by Sas2p and Sir2p functions as a shield against gene silencing; Kimura A et al.; Genes located in chromosomal regions near telomeres are transcriptionally silent, whereas those located in regions away from telomeres are not . Here we show that there is a gradient of acetylation of histone H4 at lysine 16 (H4-Lys16) along a yeast chromosome; this gradient ranges from a hypoacetylated state in regions near the telomere to a hyperacetylated state in more distant regions . The hyperacetylation is regulated by Sas2p, a member of the MYST-type family of histone acetylases, whereas hypoacetylation is under the control of Sir2p, a histone deacetylase . Loss of hyperacetylation is accompanied by an increase in localization of the telomere protein Sir3p and the inactivation of gene expression in telomere-distal regions . Thus, the Sas2p and Sir2p function in concert to regulate transcription in yeast, by acetylating and deacetylating H4-Lys16 in a mechanism that may be common to all eukaryotes. Pediatr Res, 2002 Nov, 52(5), 618 - 24 Congenital disorders of glycosylation: a review; Grunewald S et al.; Congenital disorders of glycosylation (CDGs) are a rapidly growing group of inherited disorders caused by defects in the synthesis and processing of the asparagine(ASN)-linked oligosaccharides of glycoproteins . The first CDG patients were described in 1980 . Fifteen years later, a phosphomannomutase deficiency was found as the basis of the most frequent type, CDG-Ia . In recent years several novel types have been identified . The N-glycosylation pathway is highly conserved from yeast to human, and the rapid progress in this field can largely be attributed to the systematic application of the knowledge of yeast mutants . Up to now, eight diseases have been characterized, resulting from enzyme or transport defects in the cytosol, endoplasmic reticulum, or Golgi compartment . CDGs affect all organs and particularly the CNS, except for CDG-Ib, which is mainly a hepatic-intestinal disease. Cell, 2002 Oct 18, 111(2), 173 - 84 Targeted stimulation of meiotic recombination; Pecina A et al.; Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein . DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure . We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites . At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control. Cell, 2002 Oct 18, 111(2), 163 - 72 Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p; Cheeseman IM et al.; The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known . To identify Ipl1p targets, we first purified 28 kinetochore proteins from yeast protein extracts . These studies identified five previously uncharacterized kinetochore proteins and defined two additional kinetochore subcomplexes . We then used mass spectrometry to identify 18 phosphorylation sites in 7 of these 28 proteins . Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing us to identify a consensus phosphorylation site for an Aurora kinase . Our systematic mutational analysis of the Ipl1p phosphorylation sites demonstrated that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo. Mol Cell, 2002 Sep, 10(3), 495 - 507 Multiple associated proteins regulate proteasome structure and function; Leggett DS et al.; We have identified proteins that are abundant in affinity-purified proteasomes, but absent from proteasomes as previously defined because elevated salt concentrations dissociate them during purification . The major components are a deubiquitinating enzyme (Ubp6), a ubiquitin-ligase (Hul5), and an uncharacterized protein (Ecm29) . Ecm29 tethers the proteasome core particle to the regulatory particle . Proteasome binding activates Ubp6 300-fold and is mediated by the ubiquitin-like domain of Ubp6, which is required for function in vivo . Ubp6 recognizes the proteasome base and its subunit Rpn1, suggesting that proteasome binding positions Ubp6 proximally to the substrate translocation channel . ubp6Delta mutants exhibit accelerated turnover of ubiquitin, indicating that deubiquitination events catalyzed by Ubp6 prevent translocation of ubiquitin into the proteolytic core particle. Mol Cell, 2002 Sep, 10(3), 457 - 68 Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control; Loewith R et al.; The target of rapamycin (TOR) proteins in Saccharomyces cerevisiae, TOR1 and TOR2, redundantly regulate growth in a rapamycin-sensitive manner . TOR2 additionally regulates polarization of the actin cytoskeleton in a rapamycin-insensitive manner . We describe two functionally distinct TOR complexes . TOR Complex 1 (TORC1) contains TOR1 or TOR2, KOG1 (YHR186c), and LST8 . TORC2 contains TOR2, AVO1 (YOL078w), AVO2 (YMR068w), AVO3 (YER093c), and LST8 . FKBP-rapamycin binds TORC1, and TORC1 disruption mimics rapamycin treatment, suggesting that TORC1 mediates the rapamycin-sensitive, TOR-shared pathway . FKBP-rapamycin fails to bind TORC2, and TORC2 disruption causes an actin defect, suggesting that TORC2 mediates the rapamycin-insensitive, TOR2-unique pathway . Thus, the distinct TOR complexes account for the diversity, specificity, and selective rapamycin inhibition of TOR signaling . TORC1 and possibly TORC2 are conserved from yeast to man. Mol Cell, 2002 Sep, 10(3), 441 - 2 On the road to repair: PCNA encounters SUMO and ubiquitin modifications; Matunis MJ; The molecular events and targets regulated by the RAD6 pathway, which mediates postreplication DNA repair, have remained elusive . Now, ubiquitin and SUMO modification of proliferating cell nuclear antigen (PCNA) is shown to be induced by DNA damage and linked to components of the RAD6 pathway. Dev Cell, 2002 Oct, 3(4), 467 - 8 Three-dimensional structure of a COPII prebudding complex; Barlowe C; The coat protein complex II (COPII) catalyzes transport vesicle formation from the endoplasmic reticulum . Crystallographic analysis of a Sec23/24-Sar1 prebudding complex of COPII now provides a molecular view of this GTPase-directed coat assembly mechanism. Plant Cell Physiol, 2002 Oct, 43(10), 1198 - 209 An orchid (Oncidium Gower Ramsey) AP3-like MADS gene regulates floral formation and initiation; Hsu HF et al.; cDNA for a B group MADS box gene OMADS3 was isolated and characterized from Oncidium Gower Ramsey, an important species of orchid . OMADS3 encoding a 204 amino acid protein showed high sequence homology to both paleoAP3 and TM6 lineage of B group MADS box gene such as monocots AP3 homologue LMADS1 in lily and GDEF1 in Gerbera hybrida . Despite the sequence homology, consensus motifs identified in the C-terminal region of B group genes were absent in OMADS3 . Southern analysis indicated that OMADS3 was present in O . Gower Ramsey genome in low copy numbers . Different from most B group genes, OMADS3 mRNA was detected in all four floral organs as well as in vegetative leaves . This is similar to the expression pattern of GDEF1 . 35S::OMADS3 transgenic plants showed novel phenotypes by producing terminal flowers similar to those observed in transgenic plants ectopically expressed A functional genes such as AP1 . Ectopic expression of OMADS3 cDNA truncated with the MADS box or C terminal region in Arabidopsis generated novel ap2-like flowers in which sepals and petals were converted into carpel-like and stamen-like structures . Yeast two-hybrid analysis indicated that OMADS3 is able to strongly form homodimers . Our results suggested that OMADS3 might represent an ancestral form of TM6-like gene which was conserved in monocots with a function similar to A functional gene in regulating flower formation as well as floral initiation. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14778 - 82 Epub 2002 Oct 29. Structural features of cytochrome c' folding intermediates revealed by fluorescence energy-transfer kinetics; Lee JC et al.; We employed fluorescence energy-transfer probes to investigate the polypeptide dynamics accompanying cytochrome c' folding . Analysis of fluorescence energy-transfer kinetics from wild-type Trp-72 or Trp-32 in a crystallographically characterized (1.78 A) Q1A/F32W/W72F mutant shows that there is structural heterogeneity in denatured cytochrome c' . Even at guanidine hydrochloride concentrations well beyond the unfolding transition, a substantial fraction of the polypeptides ( approximately 50%) adopts compact conformations (tryptophan-to-heme distance, approximately 25 A) in both pseudo-wild-type (Q1A) and mutant proteins . A burst phase (< or =5 ms) is revealed when stopped flow-triggered refolding is probed by tryptophan intensity: measurements on the Q1A protein show that approximately 75% of the Trp-72 fluorescence (83% for Trp-32) is quenched within the mixing deadtime, suggesting that most of the polypeptides have collapsed. J Biol Chem, 2003 Jan 3, 278(1), 147 - 53 Epub 2002 Oct 28. Pho85 phosphorylates the Glc7 protein phosphatase regulator Glc8 in vivo; Tan YS et al.; The budding yeast Glc7 serine/threonine protein phosphatase-1 is regulated by Glc8, the yeast ortholog of mammalian phosphatase inhibitor-2 . In this work, we demonstrated that similarly to inhibitor-2, Glc8 function is regulated by phosphorylation . The cyclin-dependent protein kinase, Pho85, in conjunction with the related cyclins Pcl6 and Pcl7 comprise the major Glc8 kinase in vivo and in vitro . Several glc7 mutations are dependent on the presence of Glc8 for viability . For example, glc7 alleles R121K, R142H, and R198D are lethal in combination with a glc8 deletion . We found that glc7-R121K is lethal in combination with a pho85 deletion . This finding indicates that Pho85 is the sole Glc8 kinase in vivo . Furthermore, glc7-R121K is also lethal when combined with deletions of pcl6, plc7, pcl8, and pcl10, indicating that these related cyclins redundantly activate Pho85 for Glc8 phosphorylation in vivo . In vitro kinase assays and genetic results indicate that Pho85 cyclins Pcl6 and Pcl7 comprise the predominant Glc8 kinase. Cancer Lett, 2002 Dec 15, 188(1-2), 121 - 6 Mouse DAM1 regulates pro-apoptotic activity of BLK in mammary epithelial cells; Lee S et al.; Mouse mammary gland cDNA library was screened to search for BLK-interacting protein using yeast two-hybrid system, and a mouse DNA amplified in mammary carcinoma 1 (mDAM1) was obtained . mDAM1 cDNA contained a full coding region of 678bp encoding 225 amino acids with the predicted molecular mass of 26kDa . Comparison of the mouse to human DAM1 revealed 90 and 100% identities at the nucleotide and protein levels, respectively . A single 1.5kb transcript for mDAM1 was expressed in NMuMG mouse mammary epithelial cells . Through in vitro protein binding assay, interaction between mDAM1 and BLK was also confirmed . NMuMG cells, stably transfected and expressing mDAM1, promoted cell death under serum starvation condition . Transient coexpression of mDAM1 and BLK showed increased cell death compared to BLK expression alone in NMuMG cells . These results indicate that mDAM1 promotes mammary epithelial cell death and pro-apoptotic function of BLK. Mol Microbiol, 2002 Oct, 46(2), 545 - 56 Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor; Wheeler GL et al.; Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in gamma-l-glutamyl-l-cysteinylglycine (GSH) biosynthesis, cannot grow in its absence . In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, gamma-glutamylcysteine, can partially substitute for GSH . In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH . The first contains GSH1, indicating that gamma-glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities . The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems . We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4-dependent expression of GSH1 and elevating the cellular levels of gamma-glutamylcysteine . Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4-dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression . Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule . However, this is not a general mechanism affecting all Met4-regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine . In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1-specific mechanisms exist that respond to GSH availability. Mol Microbiol, 2002 Oct, 46(2), 305 - 18 CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi; Mey G et al.; Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen . Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue . Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered . As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated . Interestingly, the C . purpurea gene, under the control of its own promoter, was able to complement the M . grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves . This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity. Chembiochem, 2002 Nov 4, 3(11), 1097 - 104 Examining reactivity and specificity of cytochrome c peroxidase by using combinatorial mutagenesis; Wilming M et al.; Combinatorial mutagenesis was used to investigate the role of three key residues in cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae, Arg48, Trp51, and Trp191, in control of the reactivity and selectivity of the heme-containing enzyme . Libraries were prepared by randomization of these residues and were subsequently screened for activity against the phenolic substrate guaiacol . Screening conditions were employed that favor either mutants with high activity or those with both high activity and stability of the reactive enzyme intermediates . The results obtained suggest a dual role for Arg48 of CCP: in addition to stabilizing reactive enzyme intermediates, the distal arginine residue plays a major role in restriction of access to the ferryl oxygen atom by small molecules and thereby controls reactivity and substrate specificity of the peroxidase . At position 51 of CCP, either a phenylalanine or a tryptophan residue is required both for catalytic and structural reasons . In contrast, either polar or positively charged residues are accepted at the position of Trp191, which is located inside the core of the protein . The variability at position 191 can be interpreted as a reflection of the mechanism of cytochrome c peroxidase, which transforms the nonpolar Trp191 into a transient cation radical. J Cell Biol, 2002 Oct 28, 159(2), 267 - 78 Epub 2002 Oct 28. Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes; Lusk CP et al.; The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown . Here, we have established a role for karyopherins in this process . We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p . This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly . At the NPC, neighboring nucleoporins bind to two regions in Nup53p . One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p . We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p . At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p . We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC. RNA, 2002 Oct, 8(10), 1334 - 47 PSF and p54nrb bind a conserved stem in U5 snRNA; Peng R et al.; PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear . Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8 . We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination . In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b . Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity . Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP. Br J Cancer, 2002 Nov 4, 87(10), 1162 - 5 Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours; Ozdag H et al.; In this study we screened the histone acetyltransferases CBP and PCAF for mutations in human epithelial cancer cell lines and primary tumours . We identified two CBP truncations (both in cell lines), seven PCAF missense variants and four CBP intronic microdeletions . These data suggest that neither gene is commonly inactivated in human epithelial cancers . Nat Cell Biol, 2002 Nov, 4(11), 880 - 7 The Doc1 subunit is a processivity factor for the anaphase-promoting complex; Carroll CW et al.; Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis . The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3) . Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction . Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner . Analysis of mutant APC lacking the Doc1/Apc10 subunit (APC(doc1 Delta)) indicates that Doc1 is required for processivity . The specific molecular defect in APC(doc1 Delta) is identified by a large increase in apparent K(M) for the cyclin substrate relative to the wild-type enzyme . This suggests that Doc1 stimulates processivity by limiting substrate dissociation . Addition of recombinant Doc1 to APC(doc1 Delta) fully restores enzyme function . Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate-enzyme affinity. J Biol Chem, 2002 Dec 27, 277(52), 50497 - 502 Epub 2002 Oct 24. Piccolo, a Ca2+ sensor in pancreatic beta-cells . Involvement of cAMP-GEFII.Rim2.Piccolo complex in cAMP-dependent exocytosis; Fujimoto K et al.; We have previously shown that cAMP-binding protein cAMP-guanidine nucleotide exchange factor II (GEFII) (or Epac2) interacting with Rim2 is involved in cAMP-dependent, protein kinase A-independent exocytosis in pancreatic beta-cells . The action of the cAMP-GEFII.Rim2 complex requires both intracellular cAMP and Ca(2+) . Although Rim2 has C(2) domains, its role as a Ca(2+) sensor has remained unclear . In the present investigation, we have discovered that Piccolo, a CAZ (cytoskeletal matrix associated with the active zone) protein in neurons that is structurally related to Rim2, also binds to cAMP-GEFII and that it forms both homodimer and heterodimer with Rim2 in a Ca(2+)-dependent manner, whereas Rim2 alone does not form the homodimer . The association of Piccolo.Rim2 heterodimerization is stronger than Piccolo.Piccolo homodimerization . Treatment of pancreatic islets with antisense oligodeoxynucleotides against Piccolo inhibits insulin secretion induced by cAMP analog 8-bromo-cyclic AMP plus high glucose stimulation . These results suggest that Piccolo serves as a Ca(2+) sensor in exocytosis in pancreatic beta-cells and that the formation of a cAMP-GEFII.Rim2.Piccolo complex is important in cAMP-induced insulin secretion . In addition, this study suggests that CAZ proteins similar to those in neurons are also function in pancreatic beta-cells. FEBS Lett, 2002 Oct 30, 531(1), 69 - 73 The structure of phosphatidylinositol transfer protein alpha reveals sites for phospholipid binding and membrane association with major implications for its function; van Tiel CM et al.; Elucidation of the three-dimensional structure of phosphatidylinositol transfer protein alpha (PI-TPalpha) void of phospholipid revealed a site of membrane association connected to a channel for phospholipid binding . Near the top of the channel specific binding sites for the phosphorylcholine and phosphorylinositol head groups were identified . The structure of this open form suggests a mechanism by which PI-TPalpha preferentially binds PI from a membrane interface . Modeling predicts that upon association of PI-TPalpha with the membrane the inositol moiety of bound PI is accessible from the medium . Upon release from the membrane PI-TPalpha adopts a closed structure with the phospholipid bound fully encapsulated . This structure provides new insights as to how PI-TPalpha may play a role in PI metabolism. Biosci Biotechnol Biochem, 2002 Sep, 66(9), 1819 - 28 CYP92B1, A cytochrome P450, expressed in petunia flower buds, that catalyzes monooxidation of long-chain fatty acids; Petkova-Andonova M et al.; In higher plants, long-chain fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds . We used the reverse trascriptase-polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida . A P450 cDNA not isolated earlier, CYP92B1, contained an open reading frame predicted to encode a polypeptide consisting of 510 amino acid residues . The transcript of the cyp92B1 gene was expressed at a high level in the early stage of flower development . CYP92B1 cDNA was expressed in a yeast, Saccharomyces cerevisiae, and recombinant yeast microsomes containing CYP92B1, a hemoprotein, metabolized lauric acid, linoleic acid, and linolenic acid. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 15154 - 8 Epub 2002 Oct 24. Neuroendocrine control of a sexually dimorphic behavior by a few neurons of the pars intercerebralis in Drosophila; Belgacem YH et al.; In Drosophila, locomotor activity is sexually dimorphic and the brain area controlling this dimorphism has been mapped . The neurons of the pars intercerebralis (PI) have been suggested to participate in such differences between males and females . However, the precise physical nature of the dimorphism, the identity of the PI neurons involved, and the nature of the neuronal signal coding the dimorphism remain unknown . In this study, we used a video-tracking paradigm to characterize further the pattern of locomotor activity in Drosophila . We show that the number of activity/inactivity periods (start/stop bouts) is also sexually dimorphic, and that it can be genetically feminized in males . Moreover, the transplantation of PI neurons from a female, or of feminized PI neurons from a donor male into a receiver wild-type male is sufficient to induce the feminization of locomotor behavior, confirming that this tiny cluster of approximately 10 neurons is directly responsible for the sexual dimorphism in locomotor activity . Finally, feeding males with fluvastatin, a juvenile hormone (JH) inhibitor, also led to start/stop feminization, and this effect is reversible by the simultaneous application of methoprene, a JH analog, suggesting the existence of a neuroendocrine control, by JH, of such behavioral dimorphism. Genetics, 2002 Oct, 162(2), 973 - 6 SIR2-induced inviability is suppressed by histone H4 overexpression; Matecic M et al.; We have identified histone H4 as a high-expression suppressor of Sir2-induced inviability in yeast cells . Overexpression of histone H3 does not suppress Sir2-induced lethality, nor does overexpression of histone H4 alleles associated with silencing defects . These results suggest a direct and specific interaction between Sir2 and H4 in the silencing mechanism. Trends Plant Sci, 2002 Oct, 7(10), 435 - 40 Homologous recombination: ends as the means; Ray A et al.; Broken chromosomal ends in somatic cells of higher plants frequently heal by the ligation of DNA ends to unrelated sequences or to sequences with micro-homologies . This pathway of DNA-strand-break repair is the bane of gene-targeting attempts in plants . However, there is a second somatic pathway of chromosome repair, which is driven by DNA-sequence homology . Observations from yeast, fly and plants of homologous-recombination mechanisms point towards new strategies of gene targeting in plants. Biochim Biophys Acta, 2002 Nov 14, 1573(2), 105 - 8 Stabilization of S-adenosyl-L-methionine promoted by trehalose; Morana A et al.; S-adenosyl-L-methionine (SAM), an important metabolic intermediate of mammals, is a well-known therapeutic agent . The molecule is chemically unstable, both in solution and in dry state, and forms different degradation products . Because the chemical instability represents a real problem during the preparation of therapeutic formulations, we investigated the capacity of some sugars to improve the SAM stability over time . In the present work, we demonstrated that the disaccharide trehalose exercises a protective effect towards the lyophilized SAM slackening its degradation (65% of SAM was detected after 50 days at 37 degrees C) . A parallel study, performed to stabilize the SAM into lyophilized yeast cells enriched in the sulfonium compound, assessed the positive effect of trehalose also in whole cells, but in lesser measure. Free Radic Biol Med, 2002 Nov 1, 33(9), 1209 - 20 Impaired mitochondrial function protects against free radical-mediated cell death; Davermann D et al.; Free radical damage can have fatal consequences . Mitochondria carry out essential cellular functions and produce high levels of reactive oxygen species (ROS) . Many agents also generate ROS . Using the yeast Saccharomyces cerevisiae as a eukaryotic model, the role of functional mitochondria in surviving free radical damage was investigated . Respiratory-deficient cells lacking mitochondrial DNA (rho(0)) were up to 100-fold more resistant than isogenic rho(+) cells to killing by ROS generated by the bleomycin-phleomycin family of oxidative agents . Up to approximately 90% of the survivors of high oxidative stress lost mitochondrial function and became "petites." The selective advantage of respiratory deficiency was studied in several strains, including DNA repair-deficient rad52/rad52 and blm5/blm5 diploid strains . These mutant strains are hypersensitive to lethal effects of free radicals and accumulate more DNA damage than related wild-type strains . Losses in mitochondrial function were dose-dependent, and mutational alteration of the RAD52 or BLM5 gene did not affect the resistance of surviving cells lacking mitochondrial function . The results indicate that inactivation of mitochondrial function protects cells against lethal effects of oxygen free radicals. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14970 - 5 Epub 2002 Oct 23. Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1; Liu C et al.; Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3' end of broken DNA . The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist . Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA . Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage . In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase . However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair . We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered. J Biol Chem, 2002 Dec 27, 277(52), 50860 - 6 Epub 2002 Oct 22. MRG15, a novel chromodomain protein, is present in two distinct multiprotein complexes involved in transcriptional activation; Pardo PS et al.; MRG15 is a novel chromodomain protein that is a member of a family of genes related to MORF4 . MORF4 (mortality factor on chromosome 4) induces senescence in a subset of human tumor cell lines . Our previous results indicated that MRG15 (MORF-related gene on chromosome 15) could derepress the B-myb promoter by association with Rb . In this study, sucrose gradient analysis demonstrated that MRG15 was present in two distinct nuclear protein complexes, MAF1 (MRG15-associated factor 1) and MAF2 . Rb was associated with MRG15 and PAM14 (a novel coil-coil protein) in MAF1, and a histone acetyl transferase, hMOF, was an MRG15 partner in MAF2 . Analysis of deletion mutants of MRG15 indicated that the leucine zipper at the C-terminal region of MRG15 was important for the protein associations in MAF1 and that the N-terminal chromodomain was required for the assembly of the MAF2 protein complex . Consistent with these data was the fact that a histone acetyltransferase activity associated with MRG15 was lost when the chromodomain was deleted and that both mutant MRG15 proteins failed to activate the B-myb promoter . The various mechanisms by which MRG15 could activate gene transcription are discussed. J Biol Chem, 2002 Dec 27, 277(52), 51017 - 24 Epub 2002 Oct 22. Functional analysis of mutations in the gamma 2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White syndrome; Daniel T et al.; Mutations in the gene encoding the gamma(2) subunit of the AMP-activated protein kinase (AMPK) have recently been shown to cause cardiac hypertrophy and ventricular pre-excitation (Wolff-Parkinson-White syndrome) . We have examined the effect of four of these mutations on AMPK activity . The mutant gamma(2) polypeptides are all able to form functional complexes following co-expression with either alpha(1)beta(1) or alpha(2)beta(1) in mammalian cells . None of the mutations caused any detectable change in the phosphorylation of threonine 172 within the alpha subunit of AMPK . Consequently, in the absence of an appropriate stimulus the mutant complexes, like the wild-type complex, exist in an inactive form demonstrating that the mutations do not lead to constitutive activation of the kinase . Three of the mutations we studied occur within the cystathionine beta-synthase (CBS) domains of gamma(2) . Two of these mutations lead to a marked decrease in AMP dependence, whereas the third reduces AMP sensitivity . These findings suggest that the CBS domains play an important role in AMP-binding within the complex . In contrast, a fourth mutation, which lies between adjacent CBS domains, has no significant effect on AMPK activity in vitro . These results indicate that mutations in gamma(2) have different effects on AMPK function, suggesting that they may lead to abnormal development of the heart through distinct mechanisms. J Biol Chem, 2002 Dec 20, 277(51), 49638 - 43 Epub 2002 Oct 22. The anti-apoptotic function of Hsp70 in the interferon-inducible double-stranded RNA-dependent protein kinase-mediated death signaling pathway requires the Fanconi anemia protein, FANCC; Pang Q et al.; Proteins encoded by five of the six known Fanconi anemia (FA) genes form a heteromeric complex that facilitates repair of DNA damage induced by cross-linking agents . A certain number of these proteins, notably FANCC, also function independently to modulate apoptotic signaling, at least in part, by suppressing ground state activation of the pro-apoptotic interferon-inducible double-stranded RNA-dependent protein kinase (PKR) . Because certain FANCC mutations interdict its anti-apoptotic function without interfering with the capacity of FANCC to participate functionally in the FA multimeric complex, we suspected that FANCC enhances cell survival independent of its participation in the complex . By investigating this function in both mammalian cells and in yeast, an organism with no FA orthologs, we show that FANCC inhibited the kinase activity of PKR both in vivo and in vitro, and this effect depended upon a physical interaction between FANCC and Hsp70 but not on interactions of FANCC with other Fanconi proteins . Hsp70, FANCC, and PKR form a ternary complex in lymphoblasts and in yeast expressing PKR . We conclude that Hsp70 requires the cooperation of FANCC to suppress PKR activity and support survival of hematopoietic cells and that FANCC does not require the multimeric FA complex to exert this function. J Biol Chem, 2002 Dec 27, 277(52), 50991 - 5 Epub 2002 Oct 22. Identification of a specific molecular repressor of the peroxisome proliferator-activated receptor gamma Coactivator-1 alpha (PGC-1alpha); Ichida M et al.; The nuclear co-activator PGC-1alpha is a pivotal regulator of numerous pathways controlling both metabolism and overall energy homeostasis . Inappropriate increases in PGC-1alpha activity have been linked to a number of pathological conditions including heart failure and diabetes mellitus . Previous studies (Puigserver, P., Adelmant, G., Wu, Z., Fan, M., Xu, J., O'Malley, B., and Spiegelman, B . M . (1999) Science 286, 1368-1371) have demonstrated an inhibitory domain within PGC-1alpha that limits transcriptional activity . Using this inhibitory domain in a yeast two-hybrid screen, we demonstrate that PGC-1alpha directly associates with the orphan nuclear receptor estrogen-related receptor-alpha (ERR-alpha) . The binding of ERR-alpha to PGC-1alpha requires the C-terminal AF2 domain of ERR-alpha . PGC-1alpha and ERR-alpha have a similar pattern of expression in human tissues, with both being present predominantly in organs with high metabolic needs such as skeletal muscle and kidney . Similarly, we show that in mice physiological stimuli such as fasting coordinately induces PGC-1alpha and ERR-alpha transcription . We also demonstrate that under normal conditions PGC-1alpha is located within discrete nuclear speckles, whereas the expression of ERR-alpha results in PGC-1alpha redistributing uniformly throughout the nucleoplasm . Finally, we show that the expression of ERR-alpha can dramatically and specifically repress PGC-1alpha transcriptional activity . These results suggest a novel mechanism of transcriptional control wherein ERR-alpha can function as a specific molecular repressor of PGC-1alpha activity . In addition, our results suggest that other co-activators might also have specific repressors, thereby identifying another layer of combinatorial complexity in transcriptional regulation. Mol Genet Genomics, 2002 Oct, 268(2), 272 - 85 Epub 2002 Sep 12. Phenotypic analysis of Paf1/RNA polymerase II complex mutations reveals connections to cell cycle regulation, protein synthesis, and lipid and nucleic acid metabolism; Betz JL et al.; Paf1 is an RNA polymerase II-associated protein in yeast, which defines a complex that is distinct from the Srb/Mediator holoenzyme . The Paf1 complex, which also contains Ctr9, Cdc73, Hpr1, Ccr4, Rtf1 and Leo1, is required for full expression of a subset of yeast genes, particularly those responsive to signals from the Pkc1/MAP kinase cascade . We have extensively characterized the pleiotropic phenotypes of deletion mutants for factors present in the Paf1 complex, identifying more than a dozen new phenotypes, and, in some cases, establishing possible molecular explanations for the growth defects . For example, paf1 Delta causes sensitivity to hydroxyurea; this phenotype correlates with a reduction in RNR1 transcript abundance and is suppressed by over-expression of RNR1 . In contrast, the resistance of paf1 Delta cells to the transcription elongation inhibitors 6-azauracil and mycophenolic acid correlates with its ability to derepress the IMD2 transcript . We tested the hypothesis that Paf1 communicates with some promoters through the DNA-binding factors Swi4, Mbp1 or Rlm1 . The phenotypes of mutations in Paf1 complex components are exacerbated in the swi4 Delta background, suggesting that the complex acts in a pathway parallel to that controlled by Swi4 . Conversely, the fact that mbp1 Delta and rlm1 Delta mutations do not enhance the phenotypes suggests that the Paf1 complex may function in the same regulatory pathway(s) with Mbp1 and Rlm1. Mol Genet Genomics, 2002 Oct, 268(2), 190 - 205 Epub 2002 Sep 20. The vesicular transport protein Cgp1p/Vps54p/Tcs3p/Luv1p is required for the integrity of the actin cytoskeleton; Fiedler TA et al.; The CGP1 gene was identified in a screen for mutations that were synthetic lethal in combination with a deletion of the gene (CPF1) for centromere and promoter factor 1 . Cells deleted for CGP1 showed reduced viability, were temperature sensitive for growth and exhibited altered sensitivity to microtubule-destabilizing drugs . Furthermore, Deltacgp1 cells showed increased rates of loss of a circular minichromosome and defects in the positioning of the short mitotic spindle . Further phenotypic analysis of Deltacgp1 cells revealed that loss of Cgp1p function led to severe depolarization of the actin cytoskeleton . In addition, cells deleted for CGP1 were hypersensitive to the actin-disrupting compound Latrunculin-A, exhibited strongly reduced polarized localization of the unconventional myosin Myo2p, and showed defects in other actin-related processes, such as shmoo formation and cell wall integrity . Cgp1p was recently identified by several groups as Vps54p, which is a member of the VFT complex that is involved in vesicular protein transport at the level of the late Golgi, acting as a tethering factor . Our data show for the first time that Cgp1p/Vps54p links aspects of vesicular protein transport with the organization of the actin cytoskeleton. Mol Genet Genomics, 2002 Oct, 268(2), 139 - 51 Epub 2002 Sep 24. clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum; Parisot D et al.; A screen for insertional mutants of Colletrichum lindemuthianum, the causative agent of common bean anthracnose, led to the identification of a non-pathogenic, lightly colored transformant . This mutant is unable to induce disease symptoms on intact or wounded primary leaves of seedlings and plantlets of Phaseolus vulgaris . In vitro, it exhibits normal vegetative growth, sporulation and conidial germination, but the cultures remain beige instead of becoming black . Microscopic examination revealed that this mutant forms fewer appressoria than the wild-type strain, and these are misshapen and poorly melanized . Molecular analyses indicated that the mutagenic plasmid had targeted clap1, a gene encoding a putative copper-transporting ATPase sharing 35% identity with the human Menkes and Wilson proteins and the product of the CCC2 gene of Saccharomyces cerevisiae . Complementation of the non-pathogenic beige mutant with a wild-type allele of clap1 restored both pathogenicity and pigmentation . Conversely, replacement of the wild-type allele with a disrupted clap1 gene gave rise to non-pathogenic beige transformants . Compared with the wild-type strain, extracts from clap1 mutants were found to have very low levels of phenol oxidase activity . These observations suggest that the clap1 gene product may be involved in the pathogenicity of C . lindemuthianum strains because of its role in delivering copper to secreted cuproenzymes, such as the phenol oxidases that mediate the polymerization of 1,8-dihydroxynaphthalene to melanin. J Biol Chem, 2002 Dec 27, 277(52), 50403 - 8 Epub 2002 Oct 21. Purification and characterization of Snf1 kinase complexes containing a defined Beta subunit composition; Nath N et al.; The Snf1 kinase complex of Saccharomyces cerevisiae contains one of three possible beta subunits encoded by either SIP1, SIP2, or GAL83 . Snf1 kinase complexes were purified from cells expressing only one of the three beta subunits using a tandem affinity purification tag on the C terminus of the Snf1 protein . The purified kinase complexes were enzymatically active as judged by their ability to phosphorylate a recombinant protein containing the Snf1-responsive domain of the Mig1 protein . The Snf1 kinase complexes containing Gal83 or Sip2 as the beta subunit showed comparable and high levels of activity, whereas the Sip1-containing enzyme was significantly less active . Examination of the protein composition of the purified Snf1 enzyme complexes indicated that the Sip1 protein was present in substoichiometric levels . Increased gene dosage of SIP1 rescued the ethanol growth defect observed in cells expressing Sip1 as their only beta subunit and increased the in vitro activity of Snf1 kinase purified from these cells . Our studies indicate that the reduced activity of Snf1-Snf4-Sip1 kinase is due to low level of Sip1 accumulation rather than a limited ability of the Sip1 form of the enzyme to direct phosphorylation of specific substrates. J Biol Chem, 2002 Dec 27, 277(52), 51033 - 42 Epub 2002 Oct 21. Phosphatidylserine transport to the mitochondria is regulated by ubiquitination; Schumacher MM et al.; Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids . Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p) . We have designed a screen for strains defective in PtdSer transport (pstA mutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Delta) genetic backgrounds . Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designate pstA1-1 . Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria . The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin, F-box) ubiquitin ligase complex . Reconstitution of different permutations of MAM and mitochondria from wild type and pstA1-1 strains demonstrates that the MET30 gene product affects both organelles . These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination. J Biol Chem, 2002 Dec 27, 277(52), 50788 - 94 Epub 2002 Oct 18. Regulation of ALK-1 signaling by the nuclear receptor LXRbeta; Mo J et al.; The transforming growth factor beta (TGF-beta) receptor, ALK-1, is expressed specifically on endothelial cells and is essential for angiogenesis, as demonstrated by its targeted deletion in mice and its mutation in the human disease hereditary hemorrhagic telangiectasia . Although ALK-1 and another endothelial-specific TGF-beta receptor, endoglin, both bind TGF-beta with identical isoform specificity and form a complex together, neither has been shown to signal in response to TGF-beta, and the mechanism by which these receptors signal in endothelial cells remains unknown . Here we report the identification of the nuclear receptor liver X receptor beta (LXRbeta) as a modulator/mediator of ALK-1 signaling . The cytoplasmic domain of ALK-1 specifically binds to LXRbeta in vitro and in vivo . Expression of activated ALK-1 results in translocation of LXRbeta from the nuclear compartment to the cytoplasmic compartment . The interaction of activated ALK-1 with LXRbeta in the cytoplasmic compartment results in the specific phosphorylation of LXRbeta by ALK-1, primarily on serine residues . LXRbeta subsequently modulates signaling by ALK-1 and the closely related TGF-beta receptor, ALK-2, as demonstrated by specific and potent inhibition of ALK-1- and ALK-2-mediated transcriptional responses, establishing LXRbeta as a potential modulator/mediator of ALK-1/ALK-2 signaling. Hum Mol Genet, 2002 Nov 1, 11(23), 2845 - 54 Filamin A and Filamin B are co-expressed within neurons during periods of neuronal migration and can physically interact; Sheen VL et al.; Mutations in the X-linked gene Filamin A (FLNA) lead to the human neurological disorder, periventricular heterotopia (PH) . Although PH is characterized by a failure in neuronal migration into the cerebral cortex with consequent formation of nodules in the ventricular and subventricular zones, many neurons appear to migrate normally, even in males, suggesting compensatory mechanisms . Here we characterize expression patterns for FlnA and a highly homologous protein Filamin B (FlnB) within the nervous system, in order to better understand their potential roles in cortical development . FlnA mRNA was widely expressed in all cortical layers while FlnB mRNA was most highly expressed in the ventricular and subventricular zones during development . In adulthood, widespread but reduced expression of FlnA and FlnB persisted throughout the cerebral cortex . FlnA and FlnB proteins were highly expressed in both the leading processes and somata of migratory neurons during corticogenesis . Postnatally, FlnA immunoreactivity was largely localized to the cell body with FlnB in the soma and neuropil during neuronal differentiation . In adulthood, diminished expression of both proteins localized to the cell soma and nucleus . Moreover, the putative FLNB homodimerization domain strongly interacted with itself or the corresponding homologous region of FLNA by yeast two-hybrid interaction, the two proteins co-localized within neuronal precursors by immunocytochemistry and the existence of FLNA-FLNB heterodimers could be detected by co-immunoprecipitation . These results suggest that FLNA and FLNB may form both homodimers and heterodimers and that their interaction could potentially compensate for the loss of FLNA function during cortical development within PH individuals. Hum Mol Genet, 2002 Nov 1, 11(23), 2815 - 28 HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis; Singaraja RR et al.; Huntington disease (HD) is caused by polyglutamine {poly(Q)} expansion in the protein huntingtin (htt) . Although the exact mechanism of disease progression remains to be elucidated, altered interactions of mutant htt with its protein partners could contribute to the disease . Using the yeast two-hybrid system, we have isolated a novel htt interacting protein, HIP14 . HIP14's interaction with htt is inversely correlated to the poly(Q) length in htt . mRNAs of 9 and 6 bp are transcribed from the HIP14 gene, with the 6 kb transcript being predominantly expressed in the brain . HIP14 protein is enriched in the brain, shows partial co-localization with htt in the striatum, and is found in medium spiny projection neurons, the subset of neurons affected in HD . HIP14 localizes to the Golgi, and to vesicles in the cytoplasm . The HIP14 protein has sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae . Expression of human HIP14 results in rescue of the temperature-sensitive lethality in akr1 Delta yeast cells and, furthermore, restores their defect in endocytosis, demonstrating a role for HIP14 in intracellular trafficking . Our findings suggest that decreased interaction between htt and HIP14 could contribute to the neuronal dysfunction in HD by perturbing normal intracellular transport pathways in neurons. EMBO Rep, 2002 Nov, 3(11), 1049 - 54 Epub 2002 Oct 22. Role of the AtRad1p endonuclease in homologous recombination in plants; Dubest S et al.; Using a specific recombination assay, we show in the plant Arabidopsis thaliana that AtRad1 protein plays a role in the removal of non-homologous tails in homologous recombination . Recombination in the presence of non-homologous overhangs is reduced 11-fold in the atrad1 mutant compared with the wild-type plants . AtRad1p is the A . thaliana homologue of the human Xpf and Saccharomyces cerevisiae Rad1 proteins . Rad1p is a subunit of the Rad1p/Rad10p structure-specific endonuclease that acts in nucleotide excision repair and inter-strand crosslink repair . This endonuclease also plays a role in mitotic recombination to remove non-homologous, 3'-ended overhangs from recombination intermediates . The Arabidopsis atrad1 mutant (uvh1), unlike rad1 mutants known from other eukaryotes, is hypersensitive to ionizing radiation . This last observation may indicate a more important role for the Rad1/Rad10 endonuclease in recombination in plants . This is the first direct demonstration of the involvement of AtRad1p in homologous recombination in plants. Eur J Biochem, 2002 Nov, 269(21), 5192 - 202 Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme; Holland L et al.; Minichromosome maintenance (MCM) proteins are part of the replication licensing factor (RLF-M), which limits the initiation of DNA replication to once per cell cycle . We have previously reported that higher order complexes of mammalian pol II and general pol II transcription factors, referred to as pol II holoenzyme, also contain MCM proteins . In the present study we have analyzed in detail the interaction between MCM2 and pol II holoenzyme . N- and C- terminal deletions were introduced into epitope-tagged MCM2 and the truncated proteins were transiently expressed in 293 cells . Affinity chromatography was used to purify RNA pol II holoenzyme and histone binding MCM complexes . We found that amino acids 168-230 of MCM2 are required for its binding to pol II holoenzyme in vivo . We also showed that bacterially expressed amino acids 169-212 of MCM2 associate with pol II and several general transcription factors in vitro . Point mutations within the 169-212 domain of MCM2 disrupted its interaction with pol II holoenzyme both in vitro and in vivo . This region is distinct from the previously characterized histone H3 binding domain of MCM2. Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14206 - 11 Epub 2002 Oct 21. Inositol pyrophosphates regulate endocytic trafficking; Saiardi A et al.; The high energy potential and rapid turnover of the recently discovered inositol pyrophosphates, such as diphosphoinositol-pentakisphosphate and bis-diphosphoinositol-tetrakisphosphate, suggest a dynamic cellular role, but no specific functions have yet been established . Using several yeast mutants with defects in inositol phosphate metabolism, we identify dramatic membrane defects selectively associated with deficient formation of inositol pyrophosphates . We show that this phenotype reflects specific abnormalities in endocytic pathways and not other components of membrane trafficking . Thus, inositol pyrophosphates are major regulators of endocytosis. Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14065 - 70 Epub 2002 Oct 21. Structure of the GCN5 histone acetyltransferase bound to a bisubstrate inhibitor; Poux AN et al.; Histone acetyltransferases (HATs) use acetyl CoA to acetylate target lysine residues within histones and other transcription factors, such as the p53 tumor suppressor, to promote gene activation . HAT enzymes fall into subfamilies